The AMPK γ1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes

Although mutations in the γ-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the γ1-subunit of AMPK, we investigated the effects of expression of an AMPK-activating γ1-subunit mutant (γ1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. Whereas expression of γ1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. In addition, glycogen synthase activity was increased in cardiac myocytes expressing γ1 R70Q, due to both increased expression and decreased phosphorylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in γ1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3β protein and mRNA levels were profoundly decreased in the γ1 R70Q-expressing myocytes. Consistent with GSK-3β negatively regulating hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3β was associated with increased nuclear activity of NFAT. Together, these data provide important new information about the mechanisms by which a mutation in the γ-subunit of AMPK causes altered AMPK signaling and identify multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the γ mutant-associated cardiomyopathy.

Muscle-Specific Deletion of Rictor Impairs Insulin-Stimulated Glucose Transport and Enhances Basal Glycogen Synthase Activity

ABSTRACT Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.